Mitochondrially targeted antioxidants

ABSTRACT

The invention provides mitochondrially targeted antioxidant compounds comprising a lipophilic cation moiety covalently coupled to a glutathione peroxidase mimetic. These compounds can be used to treat patients who would benefit from the reduction of oxidative stress.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/GB2003/003386, filed Aug. 5, 2003, and is a continuation-in-part ofU.S. patent application Ser. No. 10/217,022, filed Aug. 12, 2002 nowU.S. Pat. No. 6,984,636. This application also claims foreign prioritybenefits to Canadian Patent Application No. 2,397,684, filed Aug. 12,2002.

The present invention relates to compounds which are mitochondriallytargeted antioxidants, their use and synthesis.

Mitochondria are intracellular organelles responsible for energymetabolism. Consequently, mitochondrial defects are damaging,particularly to neural and muscle tissues which have high energydemands.

Mitochondrial dysfunction is central to a number of human degenerativediseases, and can be due to primary defects in genes encoded bymitochondrial DNA, by mutations in nuclear encoded genes, or due tosecondary consequences of other defects. Oxidative damage to themitochondrion is a major factor in the pathophysiology of thesediseases, because the mitochondrial respiratory chain is the majorsource of reactive oxygen species (ROS) within most human cells. Thesediseases include Parkinson's disease, Friedreich's Ataxia, Wilson'sDisease, mtDNA diseases, diabetes, motor neurone disease and thenon-specific loss of vigour associated with ageing. Oxidative damage tomitochondria also contributes to the pathophysiology of inflammation andischaemic-reperfusion injury in stroke, heart attack and during organtransplantation and surgery.

To prevent the damage caused by oxidative stress a number of antioxidanttherapies have been developed. In addition, a range of therapeuticallyor prophylactically useful compounds designed to protect or altermitochondrial function have been designed. The present inventors havepreviously disclosed (WO 99/26954) that certain classes of antioxidantscan be targeted to mitochondria by their covalent attachment tolipophilic cations by means of alkylene chain. In particular, thetargeting of Vitamin E and Ubiquinol to mitochondria by linking them tothe triphenyl phosphonium ion was described.

The present invention relates to the targeting of a class of antioxidantmoieties to mitochondria by their attachment to lipophilic cationmoieties.

The antioxidant moieties which have been previously targeted tomitochondria are to some extent destroyed when removing reactive oxygenspecies, in that although the active antioxidant moiety may beregenerated to some degree by processes occurring in the mitochondria orthe cell, the regeneration processes produce by-products, eventuallyleading to a complete loss or severe reduction of antioxidant function.To overcome this reduction in function, the antioxidant compounds mustbe replenished over time or be present in sufficient quantity so as toavoid a loss of efficacy of the treatment.

A first aspect of the present invention provides a compound comprising alipophilic cation moiety covalently coupled to an antioxidant moietywhich is a glutathione peroxidase mimetic.

Preferably the glutathione peroxidase mimetic is a selenoorganiccompound, i.e. an organic compound comprising at least one seleniumatom. Preferred classes of selenoorganic glutathione peroxidase mimeticsinclude benzisoselenazolones, diaryl diselenides and diaryl selenides.Such compounds, are disclosed in: Sies, H., Adv. Pharmacol., 38, 229–246(1996); Galet, V., et al., J. Med. Chem., 37, 2903–2911 (1994); Parnham,M. J., et al., Agents and Actions, 27, 306–308; Andersson, C-M., et al.,Free Radical Biol. & Med., 16, 17–28 (1994); Chaudiere, J, et al.,“Design of New Selenium-Containing Mimics of Glutathione Peroxidase”,165–184, in Oxidative Processes and Antioxidants, edited by R. Paoletti,et al., Raven Press, New York (1994), which are incorporated herein byreference.

Compounds of this aspect therefore have the following structure:

where L is a linking group and Z is an anion, and preferably apharmaceutically acceptable anion.

A second aspect of the present invention provides a compound of thefirst aspect for use in methods of treatment of the human or animalbody.

A third aspect of the present invention provides a pharmaceuticalcomposition comprising a compound of the first aspect in combinationwith one or more pharmaceutically acceptable carriers or diluents.

A fourth aspect of the present invention provides a method of treatmentof a patient who would benefit from reduced oxidative stress whichcomprises the step of administering to said patient a therapeuticallyeffective amount of a compound of the first aspect.

In a further aspect, the present invention provides a method of reducingoxidative stress in a cell which comprises the step of administering tosaid cell a compound of the first aspect.

Another aspect of the present invention provides the use of a compoundof the first aspect for the manufacture of a medicament for use in thetreatment of a condition ameliorated by reduced oxidative stress.

Conditions ameliorated by reduced oxidative stress include Parkinson'sdisease, Friedreich's Ataxia, Wilson's Disease, mtDNA diseases,diabetes, motor neurone disease, inflammation and ischaemic-reperfusiontissue injury in strokes, heart attacks, organ transplantation andsurgery.

Preferred features of the invention will be now be described by way offurther definition and example, with reference to the following figures.

FIG. 1 shows the variation in concentration of Compound 6(‘mitoebelsen’) over time in an experiment to show the take up ofcompound 6 into the mitochondrial matrix;

FIG. 2 a shows the effect on absorbance at 340 nM caused by theoxidation of NADPH in the presence of glutathione reductase and othercompounds, which were added at the time shown by the arrow: hydrogenperoxide (‘basal’); hydrogen peroxide and compound 6 (‘mitoebelsen’);and hydrogen peroxide and ebelsen (‘ebelsen’)

FIGS. 2 b–d show how the rate of NADPH oxidation varies with increasingconcentration of H₂O₂ (FIG. 2 b), GSH (FIG. 2 c), and of compound 6(‘mitoebelsen’) or ebelsen;

FIG. 3 a shows the effect on fluoresence caused by the peroxidation ofcis-parinaric acid (added at arrow) in mitochondria, in the presence ofAAPH (‘AAPH’), and how this varies in the presence of further compounds:TPMP (‘TPMP’), compound 6 (‘mitoebelsen’) and ebelsen (‘ebelsen’);

FIG. 3 b shows the same effect except in mitrochondrial membranes; and

FIG. 3 c shows the amount of MDA produced by mitochondria underoxidative stress, in the presence of compound 6 (‘mitoebelsen’), ebelsenand TPMP.

Lipophilic Cation Moieties

Mitochondria have a substantial membrane potential of up to 180 mVacross their inner membrane (negative inside). Because of thispotential, membrane permeant, lipophilic cations accumulateseveral-hundred fold within the mitochondrial matrix. (Rottenberg,Methods in Enzymology, 55, 547–560 (1979); Chen, Ann. Rev. Cell Biol.,4, 155–181 (1988)). Such ions are accumulated provided they aresufficiently lipophilic to screen the positive charge or delocalise itover a large surface area, also provided that there is no active effluxpathway and the cation is not metabolised or immediately toxic to acell.

The lipophilic cation moieties of the present invention may be ammonium,phosphonium or arsonium cations, and in particular tribenzyl ortriphenyl substituted cation moieties, of which the phenyl groups may beoptionally substituted, for example hydroxy (OH), alkoxy (O—C₁₋₇ alkyl),nitro (NO₂), amido (CONH₂), carboxy (COOH) or C₁₋₇ alkyl, at one or moreof the 3-, 4- and 5- positions. Examples include, but are not limitedto, tribenzyl ammonium, tribenzyl phosphonium, tribenzyl arsonium, andtriphenyl phosphonium cations. Of these triphenyl phosphonium ispreferred.

The lipophilic cation moieties may also be fluorescent or lightabsorbing. Examples include, but are not limited to, rhodamine 123,JC-1, N,N′-bis(2-ethyl-1,3-doxylene) kryptocyanine, pyronine Y,o-toluidine blue, chalcogenpyrilium and benzo(a)phenoxazinium (see Chen,L., Ann. Rev. Cell Biol., 4, 155–181 (1988), which is incorporatedherein by reference).

Triphenyl phosphonium is the most preferred lipophilic cation moiety forthe present invention.

Glutathione Peroxidase Mimetic Moieties

Glutathione peroxidase reduces hydrogen peroxide to water, and organichydroperoxides to alcohol, whilst oxidising glutathione to glutathionedisulphide. It is known that glutathione peroxidase has selenium as anintegral part of its active site. It is therefore preferred thatglutathione peroxidase mimetics are selenoorganic compounds, i.e.organic compounds comprising at least one selenium, atom.

A number of classes of selenoorganic glutathione peroxidase mimeticshave been disclosed in the references given above, and includebenzisoselenazolones, diaryl diselenides and diaryl selenides.

Benzisoselenazolones have the general structure:

where R can be a variety of alkyl and aryl groups. The benzene ring canbear substituents, and can also be replaced by other fused aromaticrings, for example, pyridine. The most extensively studied, andpreferred, member of this class is Ebelsen(2-phenyl-benzo[d]isoselenazol-3-one):

Diaryl diselenides are of the general formula:

where the term “aryl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 3 to 20 ring atoms (unlessotherwise specified). Preferably, each ring has from 5 to 7 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”, forexample those derived from benzene (i.e. phenyl) (C₆) and naphthalene(C₁₀), or the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”, for example those derived from pyridine(C₆), furan(C₅), thiophene (C₅) and pyrimidine (C₆).

Preferably the aryl groups are optionally substituted phenyl groups.

Examples of this class of compound include bis(2-amino)phenyl diselenideand bis(2-amino,5-acetyl)phenyl diselenide.

Diaryl selenides are of the general formula:

where the term “aryl” is as defined above. Preferably the aryl groupsare optionally substituted phenyl groups.

Examples of this class of compound include di(4-aminophenyl) selenideand di(4-phenylphenyl) selenide.

Benzisoselenazolones are the preferred class of selenoorganicglutathione peroxidase mimetics, with Ebelsen being the most preferred.

Covalent Linking Groups

The covalent linking group may be any group which joins the lipophiliccation moiety to the enzyme mimetic with a covalent bond at each end,and enables the two moieties to remain bonded together while crossingthe mitochondrial inner membrane into the mitochondrial matrix.

Typically the group will be an alkylene group. The term “alkylene,” asused herein, pertains to a bidentate moiety obtained by removing twohydrogen atoms, either both from the same carbon atom, or one from eachof two different carbon atoms, of a hydrocarbon compound having from 1to 30 carbon atoms, which may be aliphatic or alicyclic, and which maybe saturated, partially unsaturated, or fully unsaturated. Thus, theterm “alkylene” includes the sub-classes alkenylene, alkynylene,cycloalkylene, etc., discussed below.

In this context, the prefixes (e.g. C₁₋₄, C₁₋₇, C₁₋₃₀, C₂₋₇, C₃₋₇, etc.)denote the number of carbon atoms, or range of number of carbon atoms.For example, the term “C₁₋₄ alkylene” as used herein, pertains to analkylene group having from 1 to 4 carbon atoms. Examples of groups ofalkylene groups include C₁₋₄ alkylene (“lower alkylene”), C₁₋₇ alkylene,and C₁₋₃₀ alkylene.

Examples of linear saturated C₁₋₇ alkylene groups include, but are notlimited to, —(CH₂)_(n)— where n is an integer from 1 to 7, for example,—CH₂-(methylene), —CH₂CH₂-(ethylene), —CH₂CH₂CH₂-(propylene), and—CH₂CH₂CH₂CH₂—(butylene).

Examples of branched saturated C₁₋₇ alkylene groups include, but are notlimited to, —CH(CH₃)—, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—,—CH(CH₃)CH₂CH₂CH₂—, —CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—,—CH(CH₂CH₃)CH₂—, and —CH₂CH(CH₂CH₃) CH₂—.

Examples of linear partially unsaturated C₁₋₇ alkylene groups include,but are not limited to, —CH═CH-(vinylene), —CH═CH—CH₂—, —CH═CH—CH₂—CH₂—,—CH═CH—CH₂—CH₂—CH₂—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—,—CH═CH—CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH═CH—, and —CH═CH—CH₂—CH₂—CH═CH—.

Examples of branched partially unsaturated C₁₋₇ alkylene groups include,but are not limited to, —C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, and—CH═CH—CH(CH₃)—.

Examples of alicyclic saturated C₁₋₇ alkylene groups include, but arenot limited to, cyclopentylene (e.g., cyclopent-1,3-ylene), andcyclohexylene (e.g., cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₁₋₇ alkylene groupsinclude, but are not limited to, cyclopentenylene (e.g.,4-cyclopenten-1,3-ylene), cyclohexenylene (e.g., 2-cyclohexen-1,4-ylene;3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

The alkylene group may be substituted by substituent groups thatincrease the solubility of the molecule, increase the uptake of themolecule across the mitochondrial membrane, or decrease the rate ofdegradation of the molecule in vivo. In particular, the linking groupmay be substituted by: hydroxy groups, which have the formula —OH; thiogroups, which have the formula —SH; amino groups, which have the formula—NH₂; carboxy groups, which have the formula —C(═O)OH; amido groups,which have the formula —C(═O)NH₂; or groups derived from sugars or sugarderivatives.

The hydroxy, thio, amino, carboxy and amido groups are preferred locatedat the end of branches in the linking group, so that the overall effectis that of an alcohol, thiol, amine, carboxylic acid or amiderespectively attached at one end to the backbone of the linking group.

Sugars (saccharides) are carbohydrates which can be considered to behydroxylated aldehydes and ketones. If two or more monosaccharides arelinked, for example, via an acetal linkage, the compound isconventionally referred to as a disaccharide (e.g. sucrose, maltose),trisaccharide, etc., and these may all be used in the invention.Polysaccharides are not intended for use in the present invention.

Monosaccharides are conventionally named according to the overall numberof carbon atoms, for example, tri-(C₃), tetr-(C₄), pent-(C₅), andhex-(C₆). Monosaccharides may be in, for example, aldose, ketose,aldoketose, and dialdose form. Aldoses are conventionally named as -ose,for example, triose (C₃), tetrose (C₄), pentose (C₅), hexose (C₆), andheptose (C₇). Ketoses are conventionally named as -ulose, for example,tetrulose (C₄), pentulose (C₅), hexulose (C₆), and heptulose (C₇).Aldoketoses are conventionally named as -osulose. Dialdoses areconventionally named as -odialdose.

Monosaccharides may have one or more chiral centres, and thus may havedifferent stereoisomeric forms (e.g., R-, S-, D-, L-, α-, β-, (+), (−),and combinations thereof, e.g., α-D-, β-D-, α-L-, β-L-). Isomers whichare superimposable mirror images are conventionally referred to asenantiomers. Isomers which differ from each other by the configurationat two or more chiral centres are conventionally referred to asdiasteriomers. Isomers which differ from each other by the configurationat only one chiral centre are conventionally referred to as epimers(e.g., D-ribose and D-xylose).

The configuration at each chiral centre is conventionally denoted R orS. The prefixes D- or L- are conventionally used to indicatemonosaccharides with a configuration that is related to D- andL-glyceraldehyde, respectively. The prefixes (+)- and (−)- areconventionally used to indicated monosaccharides which aredextrorotatory (rotate the plane of polarised light to the right, in aclockwise direction) or levorotatory (to the left, in acounter-clockwise direction).

The prefixes erythro- and threo- denote certain tetrose (C₄)diasteriomers. The prefixes arabino-, lyxo-, ribo-, and xylo-denotecertain pentose (C₅) diasteriomers. The prefixes allo-, altro-, gluco-,manno-, gulo-, ido-, galacto-, and talo-denote certain hexose (C₆)diasteriomers.

In cyclic form (hemiacetal or hemiketal form), monosaccharides areconventionally named according to the number of ring atoms. For example,a furanose has a 5-membered ring; a pyranose has a 6-membered ring; aseptanose has a 7-membered ring. The prefixes α- and β- areconventionally used to indicate the two anomers which arise from the newchiral centre which is formed upon cyclisation.

Examples of saccharides include, but are not limited to, the following,which may be in a α-D, β-D, α-L, or β-L form:

-   -   erythrose and threose;    -   arabinose, lyxose, ribose, and xylose;    -   allose, altrose, glucose, mannose, gulose, idose, galactose, and        talose;    -   arabinofuranose, lyxofuranose, ribofuranose, and xylofuranose;    -   allofuranose, altrofuranose, glucofuranose, mannofuranose,        gulofuranose, idofuranose, qalactofuranose, talofuranose;    -   allopyranose, altropyranose, glucopyranose, mannopyranose,        gulopyranose, idopyranose, galactopyranose, and talopyranose.

Many saccharides are known by their trivial names, for example,D-threose (D-threo-tetrose), D-ribose (D-ribo-pentose), D-galactose(D-galacto-hexose), D-fructose (D-arabino-2-hexulose), L-sorbose(L-xylo-2-hexulose), D-ribulose (D-erythro-2-pentulose), D-sedoheptulose(D-altro-2-heptulose).

Many saccharides derivatives are well known, for example,deoxy-saccharides (e.g., 6-deoxy-L-galactose, also known as L-fucose;6-deoxy-L-mannose, also known as L-rhamnose; 2-deoxy-D-erythro-pentose,also known as deoxyribose or 2-deoxy-D-ribose); glycosides (e.g., methylα-D-glucopyranoside); amino-deoxy-saccharides, also known asglucosamines (e.g., D-glucosamine, D-galactosamine); alditols (e.g.,D-glutitol, also known as D-sorbitol; D-mannitol; meso-galactitol);aldonic acids, also known as glyconic acids (e.g., D-gluconic acid);uronic acids, also known as glycouronic acids (e.g., D-galactouronicacid); and aldaric acids, also known as glycaric acids (e.g.,L(+)-tartaric acid).

The alkylene group may have a heteroatom, selected from O, S or NH atits end adjacent the enzyme mimetic moiety.

Preferred linking groups are C₁₋₃₀ alkylene groups, more preferablyC₁₋₂₀, C₁₋₁₀ or C₁₋₄ alkylene groups, optionally terminating at theenzyme mimetic end with an O or S, for example —S—(CH₂)₃— and—O—(CH₂)₄—.

The linking group is preferably attached to the enzyme mimetic moiety onan aromatic ring, for example a benzene or pyridine ring.

Anions

Examples of suitable inorganic anions include, but are not limited to,those derived from the following inorganic acids: hydrochloric,hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous,phosphoric, and phosphorous

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: tetraphenylboronic,2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic,cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric,glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic,hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric,maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic,pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic,salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, andvaleric.

The above anions are generally pharmaceutically acceptable. Examples ofpharmaceutically acceptable salts are discussed in Berqe, et al.,“Pharmaceutically Acceptable Salts,” J. Pharm. Sci., 66, 1–19 (1977),which is incorporated herein by reference.

In the present invention inorganic anions are preferred, and inparticular the halo anions, of which Br⁻ is the most preferred.

Preferred Compounds

Preferred compounds of the invention have the formula:

where Z and L are as defined above, and more preferably have theformula:

where Z is as defined above, X is O, S, or NH, and preferably O or S,and n is from 1 to 20, more preferably 3 to 6.Treatment

The term “treatment” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,and cure of the condition. Treatment as a prophylactic measure (i.e.,prophylaxis) is also included.

The term “therapeutically-effective amount” as used herein, pertains tothat amount of an active compound, or a material, composition or dosageform comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio.

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously.

The compound or pharmaceutical composition comprising the compound maybe administered to a subject by any convenient route of administration,whether systemically/peripherally or topically (i.e., at the site ofdesired action).

While it is possible for the active compound to be used (e.g.,administered) alone, it is often preferable to present it as aformulation.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook for PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash),2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton,Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994,which are incorporated herein by reference.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g. human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier +which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g. liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

It will be appreciated by one of skill in the art that appropriatedosages of the compounds, and compositions comprising the compounds, canvary from patient to patient. Determining the optimal dosage willgenerally involve the balancing of the level of therapeutic benefitagainst any risk or deleterious side effects. The selected dosage levelwill depend on a variety of factors including, but not limited to, theactivity of the particular compound, the route of administration, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds, and/or materials usedin combination, the severity of the condition, and the species, sex,age, weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g. in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 1 μg to about 250 mg per kilogram body weight of the subject perday.

Further Uses

The compounds of the first and second aspects may also be useful forperfusing isolated organs prior to transport, and in storing frozencells, for example, embryos.

Synthesis Routes

Methods for the chemical synthesis of compounds of the present inventionare described herein. These methods may be modified and/or adapted inknown ways in order to facilitate the synthesis of additional compoundswithin the scope of the present invention. The amounts of reactantsgiven are for guidance. Descriptions of general laboratory methods andprocedures, useful for the preparation of the compounds of the presentinvention, are described in Vogel's Textbook of Practical OrganicChemistry (5^(th) edition, Ed. Furniss, B. S., Hannaford, A. J., Smith,P. W. G., Tatchell, A. R., Longmann, UK).

The synthesis of compounds of the present invention has three key steps:

-   (i) formation of the antioxidant moiety;-   (ii) attachment of the linking group to the lipohilic cation moiety;-   (iii)attachment of the linking group to the antioxidant moiety.

These three steps can be carried out in any order, which will bedependent on the methods used and the nature of each of the threegroups. It is possible that the formation of the antioxidant moiety canbe interrupted by linking a precursor to the linking group. Ifnecessary, protecting groups can be employed to avoid any unwantedreactions occurring during the synthesis (see Protective Groups inOrganic Synthesis, T. Green and P. Wuts; 3rd Edition; John Wiley andSons, 1999).

Formation of the antioxidant moiety: this will depend on the nature ofthe antioxidant moiety, and can usually be based on the disclosed routesfor forming that moiety. It is sometimes convenient to synthesise theenzyme mimetic moiety with the heteroatom (O, S or NH) that is on theend of the alkylene chain of the linking group adjacent the moiety, toaid the joining of the linking group to the enzyme mimetic moiety.

Linking the linking group to the lipophilic cation moiety: it isgenerally preferred to carry this step out by heating a halogenatedprecursor, preferably an iodinated or brominated precursor (RBr or RI),sometimes in an appropriate solvent with 2–3 equivalents of thelipophilic cation precursor under argon for several days. R can eitherbe the linking group, the linking group already attached to the enzymemimetic moiety, or the linking group attached to a precursor of theenzyme mimetic moiety. The compound is then isolated as its bromide oriodide salt. To do this the solvent is removed (if necessary), theproduct is then triturated repeatedly with a compound such as diethylether, until an solid remains. This can then dissolved in a solvent,e.g. dichloromethane, and precipitated with diethyl ether to remove theexcess unreacted cation. This can be repeated. Purification can involverecrystallisation, for example, from methylene chloride/diethyl ether orchromatography on silica gel eluting with dichloromethane/ethanolmixtures.

Linking the linking group to the antioxidant moiety: this will depend onthe nature of the antioxidant moiety. One method of achieving thislinking is to synthesise the linking group as part of the antioxidantmoiety. Alternatively, if the antioxidant moiety has been synthesisedwith a heteroatom in place (see above), then the linking group can bejoined by treating the antioxidant moiety with a strong base andreacting it with the linking group having a suitable leaving group (forexample, halo).

EXAMPLES General Experimental Details

Preparative column chromatography was performed using silica gel (Merck)type 60, 200–400 mesh, 40–63 um. Analytical thin layer chromatography(TLC) was performed using silica gel (Merck) 60F 254 coated on aluminaroll and visualization accomplished with UV light. Nuclear magneticresonance spectra were acquired using a Varian 300 MHz instrument.Tetramethylsilane was used as an internal standard for ¹H and ¹³C NMRexperiments in CDCl₃, and 85% phosphoric acid was used as an internalstandard for ³¹P NMR experiments in all solvents. Residual solvent peakswere used as internal standards in ¹H and ¹³C NMR experiments notperformed in CDCl₃. Chemical shift (δ) data are reported in units of ppmrelative to the internal standard. Peak assignment for ¹³C NMR were madeon the basis of chemical shift, relative intensity and HSQC data.Infrared absorption spectra were acquired using a Perkin Elmer SpectrumBX FTIR instrument. Samples were examined as KBr discs prepared usinganhydrous KBr and units of absorption are reported in wavenumbers(cm⁻¹). Low resolution electrospray (LRES) and low resolutionatmospheric pressure chemical ionisation (LRAPCI) mass spectra wereacquired using a Shimadzu LCMS-QP800X liquid chromatograph massspectrometer and data are reported as m/z values. Melting points wereacquired using a Kofler Heizbank melting point bench and are reporteduncorrected.

Synthesis of2-[4-(4-triphenylphosphoniobutoxy)phenyl]-1,2-benzisoselenazol)-3(2H)-oneiodide (Compound 6)

N-(4-hydroxyphenyl)-benzamide (Compound 1)

A solution of benzoyl chloride (12.9 g, 91.7 mmol) in dry THF (250 mL)was added dropwise to a solution of 4-aminophenol (10.0 g, 91.7 mmol)and Et₃N (9.4 g, 92.9 mmol) in dry THF (700 mL), and the mixture stirredfor 2 days under a drying tube. Solvent was removed in vacuo and thesolid residue extracted with H₂O (3×250 mL) and diethyl ether (3×250mL). The remaining solid material was recrystallised from aqueousethanol, the crystals filtered, washed with diethyl ether and suckeddry, giving 1 as white crystals (14.97 g, 77%). ¹H NMR (299.9 MHz,d₆-DMSO) δ 10.06 (broad s, 1H, —OH), 9.28 (s, 1H, —NH—), 7.96 (d, J=7.8Hz, 2H, Ar—H), 7.57 (m, 5, Ar—H), 6.78 (d, J=7.8 Hz, Ar—H); ¹³C NMR(125.7 MHz, d₆-DMSO) δ 165.92 (carbonyl), 154.56 (aromatic), 136.09(aromatic), 132.30 (aromatic), 131.61 (aromatic), 129.33 (protonatedaromatic), 128.48 (protonated aromatic), 123.21 (protonated aromatic),115.90 (protonated aromatic); Anal. Calcd. for C₁₃H₁₁NO₂: C,73.22; H,5.20; N; 6.57. found: C, 73.33; H, 5.27, N, 6.69; m.p. 224° C.

N-[4-(tert-Butyldimethylsiloxy)phenyl]benzamide (Compound 2)

A solution of 1 (14.5 g, 68.01 mmol) and imidazole (11.6 g, 67.91 mmol)in dry THF (160 mL) was cooled to 0° C. under a drying tube. A solutionof TBDMSCl in dry THF (60 mL) was added dropwise under a drying tube tothe cooled solution. The resultant suspension was stirred overnightunder an argon atmosphere. The suspension was then diluted with H₂O (500mL) and extracted with CH₂Cl₂ (2×500 mL). The organic extracts werecombined, dried over Na₂SO₄, and filtered. Solvent was removed from thefiltrate in vacuo, giving a white solid. The solid was recrystallisedfrom hexane after a hot filtration. The crystals were collected byfiltration, washed with hexane and sucked dry, giving sufficiently pure2 as white crystals (19.86 g, 89%); ¹H NMR (299.9 MHz, CDCl₃) δ 7.86 (d,J=8.1 Hz, 1H, Ar—H), 7.85 (d, J=8.1 Hz, 1H, Ar—H), 7.57–7.44 (m, 5H,Ar—H), 6.84 (d, J=8.5 Hz, 2H, Ar—H), 0.99 (s, 9H, t-butyl methyl), 0.20(s, 6H, methyl); ¹³CNMR (125.7 MHz, CDCl₃) δ 165.97 (carbonyl), 152.9(aromatic), 135.45 (aromatic), 132.04 (protonated aromatic), 131.88(aromatic), 129.11 (protonated aromatic), 127.31 (protonated aromatic),122.22 (protonated aromatic), 120.80 (protonated aromatic), 26.04(methyl), 18.56 (tertiary), −4.10 (methyl); LRAPCI MS (+formic acid)Calcd. For C₁₉H₂₄NO₂SiSe; 328. found: 328.

2-[4-(tert-Butyldimethylsiloxy)phenyl]-1,2-benzisoselenazol-3(2H)-one(Compound 3)

To a solution of 2 (9.20 g, 28.09 mmol) in dry THF (150 mL) under argonand cooled to −15° C. was slowly added n-BuLi (1.6 M in hexane, 35 mL,56.0 mmol) via a cannula over 45 minutes. The resultant orange solutionwas stirred at −15° C. for 45 minutes and selenium powder (2.22 g, 28.12mmol) was then added. The resultant suspension was stirred for 45minutes at −15° C., giving a deep red solution. The solution was cooledto −78° C. and CuBr₂ (12.55 g, 56.18 mmol) was added in 3 portions over15 minutes. The resultant suspension was stirred for 1h at −78° C. thenremoved from the cooling bath and stirred for 22 hours. The resultantbrown suspension was poured into 1% aqueous acetic acid (600 mL) andextracted with CH₂Cl₂ (3×500 mL). The organic fractions were combinedand filtered. The filtrate was dried over Na₂SO₄, filtered and solventremoved from the filtrate in vacuo, to give a brown greasy solid. Thesolid was chromatographed (silica gel packed in CH₂Cl₂, eluting withCH₂Cl₂), fractions containing product were combined and solvent removedin vacuo, giving a pale tan solid. The solid was recrystallised fromEtOH, the crystals filtered, washed with hexane and sucked dry, giving 3as pale yellow crystals (5.09 g, 45%). ¹H NMR (299.9 MHz, CDCl₃) δ 8.11(d, J=7.8 Hz, 1H Ar—H), 7.64–7.60 (m, 2H, Ar—H), 7.50–7.42 (m, 3H,Ar—H), 6.88 (d, J=9 Hz, 2H, Ar—H), 1.00 (s, 9H, t-butyl methyl), 0.22(s, 6H, methyl); ¹³C NMR (125.7 MHz) δ 166.12 (carbonyl), 154.91(aromatic), 138.09 (aromatic), 132.72 (protonated aromatic), 132.54(aromatic) 129.72 (protonated aromatic), 127.78 (aromatic), 127.45(protonated aromatic), 126.82 (protonated aromatic), 124.05 (protonatedaromatic), 120.96 (protonated aromatic), 25.71 (t-butyl methyl), 18.27(tertiary), −4.34 (methyl); Anal Calcd. for C₁₉H₂₃NO₂SiSe: C, 56.43, H,5.73, N, 3.46. found: C, 56.72, H, 5.84, N, 3.56; m.p. 188° C.

2-(4-Hydroxyphenyl)-1,2-benzisoselenazol-3(2H)-one (Compound 4)

To a 0° C. solution of 3 (2.0 g, 4.95 mmol) in THF (25 mL) was addedTBAF solution (1M in THF, 14 mL, 14 mmol) dropwise over 15 minutes undera nitrogen atmosphere. The mixture was stirred at 0° C. for 1 hour afterwhich time TLC analysis (silica gel, 19:1 CH2Cl2/diethyl ether) showedno starting material to be present. The solvent was removed in vacuo togive a brown oil. The oil was dissolved in CH₂Cl₂ (50 mL) and theorganic phase washed with 5% HCl (150 mL), and H₂O (4×50 mL). Upon thesecond water wash a yellow solid formed inside the separating funnel.The solid was collected by filtering both phases and residual solid waswashed from the funnel in the final two washes. The filtered solid waswashed with the final two aqueous extracts, sucked dry andrecrystallised from 1:1 EtOH/THF (35 mL). The crystals were collected byfiltration, washed with diethyl ether and sucked dry, giving 4 as yellowcrystals (1.01 g, 97%). ¹H NMR (299.9 MHz, d₆-DMSO) δ 9.65 (s, 1H, —OH),8.10 (d, J=7.8 Hz, 1H, Ar—H), 7.91 (d, J=8.4 Hz, Ar—H), 7.70 (dd, 1H,Ar—H), 7.49 (dd, 1H, Ar—H), 7.39 (d, J=9 Hz, 2H, Ar—H), 6.86 (d, J=9 Hz,2H, Ar—H); ¹³C NMR (125.7 MHz, d₆-DMSO) δ 165.82 (carbonyl), 156.71(aromatic), 139.94 (aromatic), 132.87 (aromatic), 131.54 (aromatic),129.26 (aromatic), 128.75 (aromatic), 127.70 (protonated aromatic),127.07 (aromatic), 126.71 (aromatic), 116.51 (protonated aromatic); AnalCalcd. for C₁₃H₉NO₂Se: C, 53.81, H, 3.13, N, 4.83. found: C,54.04, H,3.03, N, 4.88; m.p. >260° C.

2-[4-(4-iodobutoxy)phenyl]-1,2-benzisoselenazol-3(2H)-one (Compound 5)

To a suspension of NaH (60% emulsion washed with pentane and dried invacuo, 60 mg, 1.50 mmol) in dry DMF (1 mL) at 0° C. under argon wasadded a solution of 4 (300 mg, 1.03 mmol) in dry DMF (7 mL) under argonvia a cannula. The resultant solution was stirred for 2 hours at roomtemperature then added via a cannula to a solution of 1,4-diiodobutane(3.20 g, 10.3 mmol) in dry DMF (2 mL) under argon. The resultantsolution was stirred in the dark for 2 days at room temperature underargon. Water (1 mL) was carefully added and solvent removed in vacuo togive an oily residue. The residue was dissolved in CH₂Cl₂ (20 mL) andthe organic phase washed with H₂O (20 mL), 10% Na₂S₂O₃ (20 mL), and H₂O(20 mL). The organic extract was dried over Na₂SO₄ and filtered. Solventwas removed from the filtrate in vacuo, giving a yellow oily residue.The residue was chromatographed (silica gel packed in CH₂Cl₂, elutingwith 19:1 CH₂Cl₂/diethyl ether) and fractions containing product werecombined. Solvent was removed in vacuo, giving sufficiently pure 5 as awhite solid (254 mg, 52%). ¹H NMR (299.9 MHz, CDCl₃) δ 8.11 (d, J=7.8Hz, 2H, Ar—H), 7.68–7.62 (m, 2H, Ar—H), 7.52–7.43 (m, 3H, Ar—H), 6.94(d, J=9 Hz, 2H, Ar—H), 4.02 (t, J=5.9 Hz, 2H, —O—CH₂—), 3.27 (t, J=6.6Hz, 2H, —CH₂—I), 1.98 (m, 4H, —CH₂—CH₂—); ¹³CNMR (125.7 MHz, CDCl₃) δ166.20 (carbonyl), 158.0 (aromatic), 138.12 (aromatic), 132.73(aromatic), 132.03 (aromatic), 129.73 (aromatic), 127.73 (protonatedaromatic), 127.60 (aromatic), 126.85 (aromatic), 126.85 (aromatic),124.08 (aromatic), 115.41 (protonated aromatic), 67.32 (—O—CH₂—), 30.43(methylene), 6.64 (—CH₂—I); LRAPCI MS (+formic acid) Calcd. ForC₁₇H₁₇NOSeI: 473. found: 473.

2-[4-(4-triphenylphosphoniobutoxy)phenyl]-1,2-benzisoselenazol)-3(2H)-oneiodide (Compound 6)

A dry Kimax tube containing 5 (50 mg, 0.106 mmol) and triphenylphosphine(278 mg, 1.06 mmol) was purged with argon, sealed and placed in thedark. The mixture was stirred as a melt at 90° C. for 2 hours. Themixture was cooled and the solid residue dissolved in CH₂Cl₂ (1 mL).Product was triturated with diethyl ether and collected bycentrifugation (3500 rpm for 10 minutes). The solid residue wastriturated twice more from CH₂Cl₂ with diethyl ether and collected bycentrifugation. Residual solvent was removed from the precipitate invacuo, giving crude 6 as a yellow solid (68 mg, 87%). ¹H NMR (299.9 MHz,CD₂Cl₂) δ 8.44 (d, J=8.1 Hz, 1H, Ar—H), 7.90 (d, J=8 Hz, 1H, Ar—H),7.86–7.34 (m, 19H, Ar—H, —P⁺Ph₃), 6.79 (d, J=9 Hz, 2H, Ar—H), 3.97 (t,J=5.7 Hz, 2H, —O—CH₂—), 3.41 (m, 2H, —CH₂—P⁺Ph₃), 2.04 (m, 2H,—O—CH₂—CH₂—), 1.87 (m, 2H, —CH₂—CH₂—P⁺Ph₃); ³¹P NMR (121.4 MHz, CD₂Cl₂)δ 24.29; LRES⁺ Calcd for [C₃₅H₃₁NO₂PSe]⁺: 608. found: 608

Accumulation of Compound 6 in Mitochondria

To demonstrate that the targeting of the glutathione peroxidase mimeticto mitochondria is effective and that these compounds can cross thelipid bilayer of the mitochondrial inner membrane, compound 6 was testedin relation to isolated mitochondria.

Liver mitochondria were prepared from female Wistar rats byhomogenisation followed by differential centrifugation in mediumcontaining 250 mM sucrose, 10 mM Tris-HCL (pH 7.4) and 1 mM EGTA(Chappell, J. B. and Hansford, R. G., In: Subcellular components:preparation and fractionation (Ed. Birnie G D), pp. 77–91. Butterworths,London, 1972, which is incorporated herein by reference.)

An ion selective electrode for compound 6 was prepared by standardprocedures (Kamo, N., et al., Journal of Membrane Biology, 49, 105–121(1979) and Davey, G. P., et al., Biochemical Journal, 288, 439–443(1992), which are incorporated herein by reference), and activated bysoaking in 100 μM compound 6 for 1–3 days.

The rat liver mitochondria (0.5 mg protein/ml) were suspended in 2 mLKCl medium (120 mM KCl, 10 mM HEPES, pH 7.2, 1 mM EGTA) with additionsof rotenone (2 μg/ml) in a 30° C. thermostatted chamber. Compound 6 wastitrated in in 1 μM increments, as 1.5 μl aliquots in DMSO, and theresponse of the electrode measured over 0–5 μM of Compound 6. Thisresponse was logarithmic with respect to the concentration of Compound6.

To energise the mitochondria, the respiratory substrate succinate (10mM) was added. These conditions are known to generate a mitochondrialmembrane potential of about 180 mV (Burns, et al., Arch Biochem Biophys,332, 60–68 (1995); Burns and Murphy, Arch Biochem Biophys, 339, 33–39(1997), which are incorporated herein by reference). As can be seen fromFIG. 1, the concentration of compound 6 outside the mitochondria fallsrapidly.

When the uncoupler FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone), which prevents mitochondriaestablishing a membrane potential (Burns et al., 1995))(332 nM) wasadded to dissipate the membrane potential, the accumulation of compound6 into mitochondria was reversed, as seen in FIG. 1.

This shows that compound 6 is rapidly and selectively accumulated intomitochondria driven by the mitochondrial membrane potential. As thisaccumulation is rapidly (<10 s) reversed by addition of the uncouplerFCCP to dissipate the mitochondrial membrane potential afteraccumulation of compound 6 within the mitochondria, the mitochondrialspecific accumulation of compound 6 is solely due to the mitochondrialmembrane potential and is not due to specific binding or covalentinteraction.

Glutathione Peroxidase Assay

The activity of the glutathione peroxidase mimetics was determinedindirectly by measuring the stimulated oxidation of NADPH in thepresence of glutathione reductase. The incubations were carried out at37° C. in quartz cuvettes containing 143 mM potassium phosphate buffer,6.3 mM EDTA, pH 7.5, 250 μM NADPH, 1 mM GSH, 50 μM ebselen or compound 6and 1 U glutathione reductase. The reaction was started with 1 mMhydrogen peroxide or 1 mM tert butyl hydroperoxide and was recorded at340 nm with air as a reference by a Shimadzu model UV-2501PCSpectrophotometer. The reaction was further tested with increasingconcentrations of ebselen and compound 6, glutathione and oxidants.

The indirect glutathione peroxidase assay indicates that the basaloxidation of GSH by hydrogen peroxide amounts to 55±2 nmol NADPH/min incontrol incubations without glutathione peroxidase mimetics. This basalrate was accelerated three fold by Ebselen giving a rate of 140±4 nmolNADPH/min. Compound 6 had about two fold increase over the basaloxidation with a rate of 115±4 nmol NADPH/min (FIG. 2 a). The basaloxidation of GSH by t-butyl hydroperoxide was 29 ±1 nmol NADPH/min andthis was increased two fold by compound 6 and three fold by Ebselen(data not shown). This NADPH oxidation is accelerated by increasing theconcentrations of hydroperoxides (FIG. 2 b), GSH (FIG. 2 c) and compound6 or ebselen (FIG. 2 d). No reaction was observed in the absence of GSHand peroxide. This confirms that compound 6 is an effective mimetic.

Antioxidant Efficacy of Compound 6

To determine the anioxidant efficacy of compound 6, mitochondria ormitochondrial membranes were incubated with cis-parinaric acid. Thisfatty acid fluoresces when incorporated into lipid bilayers. Uponoxidation of its conjugated double bond fluorophore the flurescence isdecreased and this is a measure of lipid peroxidation.

To measure cis-parinaric acid oxidation, rat liver mitochondria (0.5 mgprotein/mL) were suspended in a 3 mL fluorometer cuvette in 100 mM KCland 10 mM Tris-HCl, pH 7.6, 10 mM succinate, 8 μg/mL rotenone, 10 mM2,2′—azobis(amidinopropane) dihydrochloride (AAPH) in the presence orabsence of 1 μM ebselen or compound 6 (‘mitoebelsen’) at 37° C. Controlincubations were in the presence of 1 μM TPMP. The oxidation reactionwas started with the addition of 0.5 μM cis-parinaric acid and thefluorescence was measured λ_(excite)=324 nm, λ_(emission)=413 nm).Control incubation of cis-parinaric acid into a mitochondrial suspensionshowed a slow decay of fluorescence over time due to self quenching.Cis-parinaric acid oxidation was measured using beef heart mitochondrialmembranes (0.1 mg protein/mL) in 50 mM potassium phosphate buffer, pH8.0 using the same conditions described above.

In the presence of mitochondria or mitochondrial membranes cis-parinaricacid fluoresces and has a slow autooxidation rate (FIG. 3 a). This basaloxidation rate is rapidly accelerated in the presence of the radicalinitiator of lipid peroxidation AAPH (FIG. 3 a). In mitochondria 1 μMcompound 6 (‘mitoebselen’) prevented the oxidation of cis-parinaric acidby AAPH, showing its antioxidant activity, and ebselen at thisconcentration failed to protect cis-parinaric acid from oxidation (FIG.3 a). Control incubation with TPMP did not slow down the oxidation ofthe cis-parinaric acid (FIG. 3 a). Similar experiments withmitochondrial membranes showed that 5 μM compound 6 (‘mitoebselen’) andebselen effectively protect against oxidation of cis-parinaric acid(FIG. 3 b). The lipophilic cation TPMP has no effect on cis-parinaricoxidation confirming the antioxidant effect of compound 6(‘mitoebselen’) and ebselen (FIG. 3 b).

To quantitate the antoxidant effect of compound 6, mitochondria wereincubated with rat liver mitochondria (2 mg protein/mL) at 37° C. for 15min in 100 mM KCl and 10 mM Tris-HCl, pH 7.6, 10 mM succinate, 8 μg/mLrotenone, 50 μM FeCl2 in the presence or absence of 1 μM compound 6 orebselen. Control incubations were in the presence of 1 μM TPMP and FeCl₂alone. Then 0.8 mL aliquots were mixed with, 0.1%, butylatedhydroxytoluene, 200 μL of 1% thiobarbituric acid and 200 μL 35% HClO₄,heated at 100° C. fro 15 min, diluted with 3 mL water, and extractedinto 3 mL n-butanol. TBARS (thiobarbituric acid relative species) weredetermined fluorometrically (λ_(excite)=515 nm, λ_(emission)=553 nm) ina plate reader and expressed as nanomoles of MDA by comparison withstandard solutions of 1,1,3,3-tetraethoxypropane processed as above, andthe accumulation of MDA was measured as a marker of lipid peroxidation.

Incubation of mitochondria with 1 μM compound 6 (‘mitoebselen’)prevented the accumulation of MDA (FIG. 3 c) Control incubations with 1μM TPMP did not prevent MDA accumulation confirming that the antioxidanteffect of compound 6 is due to its ebselen selenol moiety (FIG. 3 c).

1. A compound comprising a lipophilic cation moiety, wherein the lipophilic cation moiety is selected from the group consisting of: ammonium cations; phosphonium cations; arsonium cations; rhodamine 123; JC-1; N,N′-bis(2-ethyl-1,3-doxylene)kryptocyanine; pyronine Y; o-toluidine blue; chalcogenpyrilium; benzo(a)phenoxazinium and a tribenzyl or triphenyl substituted ammonium or phosphonium cation, covalently coupled to an antioxidant moiety which is a glutathione peroxidase mimetic, wherein the glutathione peroxidase mimetic moiety is selected from the group consisting of: benzisoselenazolones, diaryl diselenides and diaryl selenides.
 2. A compound according to claim 1, wherein the glutathione peroxidase mimetic moiety comprises at least one selenium atom.
 3. A compound according to claim 1, wherein the glutathione peroxidase mimetic moiety is:


4. A compound according to claim 1, wherein the lipophilic cation moiety is triphenyl phosphonium.
 5. A compound according to claim 1 having the formula:

where L is a linking group and Z is an anion.
 6. A compound according to claim 5, wherein the linking group is an alkylene group having 1 to 30 carbon atoms, optionally comprising O, S or NH at the glutathione peroxidase mimetic moiety end, and is optionally substituted by a group selected from: hydroxy, thio, amino, carboxy, amido and sugar derivatives.
 7. A compound according to claim 5, wherein Z is a pharmaceutically acceptable anion.
 8. A compound according to claim 5, wherein the compound is of formula:


9. A compound according to claim 8, wherein the compound is of formula:

where X is selected from the group consisting of: O, S, and n is from 1 to
 30. 10. A pharmaceutical composition comprising a compound according to claim 1 in combination with one or more pharmaceutically acceptable carriers or diluents.
 11. A method of reducing oxidative stress in a human or animal which comprises the step of administering to the human or animal a therapeutically effective amount of a compound according to claim
 1. 12. The method according to claim 11, wherein the human is suffering from a condition selected from Parkinson's disease, Friedreich's Ataxia, Wilson's Disease, mtDNA diseases, diabetes, motor neurone disease, inflammation and ischaemic-reperfusion tissue injury in strokes, heart attacks, organ transplantation and surgery. 